PROJECT SUMMARY Rhabdoid tumor (RT) is a pediatric malignancy characterized by a single recurrent coding mutation in the chromatin remodeling complex, SWI/SNF, leading to overactivation of polycomb repressive complex 2 (PRC2). In normal cells these complexes oppose each other, however in RT, the defect in SWI/SNF and activation of PRC2 establishes an imbalance that drives malignant transformation and progression. Therefore, the dysregulated balance between SWI/SNF and PRC2 is widely regarded as the therapeutic vulnerability in rhabdoid tumor. Our lab identified a unique sensitivity of rhabdoid tumor to the transcriptional inhibitor, mithramycin. Due to the tumor?s dependence on SWI/SNF and PRC2, the overall hypothesis of this proposal is that rhabdoid tumor sensitivity to mithramycin is due to disruption of the SWI/SNF-PRC2 axis. Mechanistically, the SWI/SNF-PRC2 balance maintains a proliferative and pluripotent cellular status suggesting modulation of this balance may lead to apoptosis or differentiation. Therefore, the goal of this study is to define the optimal therapeutic endpoint of mithramycin in rhabdoid tumor. In aim 1, we will elucidate the mechanism of rhabdoid tumor sensitivity to mithramycin. By understanding the mechanism of mithramycin in RT, we can validate mithramycin as a targeted therapy for rhabdoid tumor. Our preliminary data indicates mithramycin can induce both an apoptotic and differentiation phenotype depending on dose and exposure of the drug. We will determine the transcriptional network responsible for the divergent phenotypes and correlate the phenotypes with SWI/SNF-PRC2 dynamics. In aim 2.1, we will use second-generation analogues of mithramycin as chemical probes to maximize the clinical potential of this compound. In this aim, we will develop more potent and less toxic compounds that increase the penetrance of apoptosis and differentiation endpoints. We will determine the mechanism responsible for their improved toxicity profile and directly compare the therapeutic endpoints of apoptosis and differentiation in vitro and in vivo. In aim 2.2, we will employ an unbiased screening approach to identify additional therapeutic targets in rhabdoid tumor that cooperate with or are independent of SWI/SNF and PRC2. We will define novel differentiation targets that are dependent on SMARCB1-deficiency and therefore specific to rhabdoid tumor. Further, we will define targets that are dependent on SMARCB1 and mithramycin and therefore, are candidates for combination therapy development. In summary, this study addresses the need for a targeted therapy in rhabdoid tumor. Specifically, we will characterize apoptotic and differentiation endpoints by targeting the transcription of epigenetic modifiers, thereby defining a novel approach to targeting epigenetics. Further, we will characterize the optimal therapeutic endpoint for rhabdoid tumor to inform the clinical translation of the therapies developed in this proposal.